The cloning of a platelet thrombin receptor has provided a framework to understand how thrombin interacts with cells, and has suggested a new target for antithrombotic and other therapies (Coughlin, S. R.; Scarborough, R. M.; Vu, T.-K. H.; Hung, D. T. (1992) Thrombin Receptor Structure and Function. Cold Spring Harbor Symposia on Quantitative Biology 57, 149-154; Coughlin, S. R.; Vu, T.-K. H.; Hung, D. T.; Wheaton, V. I. (1992) Expression Cloning and Characterization of a Functional Thrombin Receptor Structure Reveals a Novel Proteolytic Mechanism of Receptor Activation. Semin. Thromb. Haemostasis 18, 161-166). The first thrombin receptor to be cloned and sequenced was that from a human megakaryocytoblastoma cell line (Vu et al., 1991, infra). This human "platelet" receptor was expressed in different cell types, such as Chinese hamster ovary (CHO) cells, and appears capable of mediating standard cellular responses to thrombin (calcium flux, phosphoinositide turnover, cell proliferation). The thrombin receptor has also been cloned and sequenced for other mammalian cell types. Amino acid sequences of functional thrombin receptors from human platelets (Vu, T.-K. H., Hung, D. T., Wheaton, V. I., Coughlin, S. R. (1991) Molecular Cloning of the Functional Thrombin Receptor Reveals a Novel Proteolytic Mechanism of Receptor Activation. Cell 64, 1057-1068.), Chinese hamster lung fibroblasts (Rasmussen, U. B., et al. (1991) cDNA Cloning and Expression of a Hamster a-Thrombin Receptor Coupled to Ca.sup.2+ Mobilization. FEBS Lett., 288, 123-128), rat vascular smooth muscle cell (Zhong, C.; Hayzer, D. J.; Corson, M. A.; Runge, M. S. (1992) Molecular Cloning of the Rat Vascular Smooth Muscle Thrombin Receptor. Evidence for In Vitro Regulation by Basic Fibroblast Growth Factor. J. Biol. Chem. 267, 16975-16979), and mouse osteoblastic cells (Tanaka, H.; Suva, L. J.; Suong, L. T.; Rodan, G. A. (1993) Cloning of the Mouse Thrombin Receptor from Osteoblastic Cells and Regulation of its Expression by 1,25-Dihydroxyvitamin D.sub.3 and Parathyroid Hormone. J. Bone Mineral Res. Abstr. 108.) were derived from cDNA cloning and found to have a high degree of homology.
Structural analysis of the protein sequence revealed that the thrombin receptor is a member of the G-protein coupled receptor (GPCR) superfamily, with seven transmembrane (TM) domains, an extracellular amino terminus, and a cytoplasmic carboxy terminus. A guanine nucleotide-binding protein, which is key to cytoplasmic signal transduction, probably associates with intracellular loops 2 and 3, and the carboxy terminus. Thrombin proteolytically cleaves the long extracellular amino terminus between Arg-41 and Ser-42 to expose a new amino terminus which functions as a tethered peptide ligand for a yet-unknown recognition domain in the body of the receptor that induces receptor activation (Vu, T.-K. H.; Hung, D. T.; Wheaton, V. I.; Coughlin, S. R. (1991) Molecular Cloning of the Functional Thrombin Receptor Reveals a Novel Proteolytic Mechanism of Receptor Activation. Cell 64, 1057-1068). Thus the thrombin receptor is unique among GPCR's in that its activating ligand is self-contained, rather than generated separately as a hormone or transmitter.
The model of receptor-thrombin interaction, receptor cleavage, and signal transduction has been supported by studies with structural variants of the receptor and specific peptides (Vu, T.-K. H.; Wheaton, V. I.; Hung, D. T.; Charo, I.; Coughlin, S. R. (1991) Domians Specifying Thrombin-Receptor Interaction. Nature 353, 674-677), and monoclonal antibodies (Brass, L. F. (1992) Homologous Desensitization of HEL Cell Thrombin Receptors. J. Biol. Chem. 267, 6044-6050; Bahou, W. F.; Coller, B. SW.; Potter, C. L.; Norton, K. J.; Kutok, J. L.; Goligorsky, M. S. (1993) The Thrombin Receptor Extracellular Domain Contains Sites Crucial for Peptide-Ligand-Induced Activation. J. Clin. Invest. 91, 1405-1413; Norton, K. J.; Scarborough, R. M.; Kutok, J. L.; Escobedo, M.-A.; Nannizzi, L.; Coller, B. S. (1993) Immunologic Analysis of the Cloned Platelet Thrombin Receptor Activation Mechanism: Evidence Supporting Receptor Cleavage, Release of the N-Termianl Peptide, and Insertion of the Tethered Ligand into a Protected Environment. Blood 82, 2125-2136). Studies with mutant thrombin receptors have shown that cleavage of the N-terminus of the receptor is necessary for activation. Mutations in the tethered ligand domain inhibit activation of the expressed receptor (Scarborough, R. M., et al. (1992) Tethered Ligand Agonist Peptides: Structural Requirments for Thrombin Receptor Activation Reveal Mechanism of Proteolytic Unmasking of Agonist Function. J. Biol. Chem. 267, 13146-13149).
The unique mechanism for proteolytic receptor activation also raises a long standing question about thrombin-cell interaction. How are thrombin cellular responses elicited in a classical, concentration-dependent, ligand-receptor mechanism rather than through enzyme-based activity? In fact, the rate of receptor cleavage is proportional to thrombin concentration (Hung, D. T.; Vu, T.-K. H.; Nelken, N. A.; Coughlin, S. R. (1992) Thrombin-Induced Events in Non-Platelet Cells are Mediated by the Unique Proteolytic Mechanism Established for the Platelet Thrombin Receptor. J. Cell Biol. 116, 827-832.). However, low concentrations of thrombin ultimately cleave and activate all thrombin receptors. Hence, a novel shut-off mechanism to deal with the "irreversibility" nature of the tethered ligand must exist within the cell, in particular, because shut-off can occur despite the continued presence of cleaved/activated receptor. Cumulative phosphoinositide hydrolysis in response to thrombin correlates precisely with cumulative receptor cleavage as a function of time (Ishii, K.; Hein, L.; Kobilka, B.; Coughlin, S. R. (1993) Kinetics of Thrombin Receptor Cleavage on Intact Cells. Relation to Signaling. J. Biol. Chem. 268, 9780-9786). These data strongly suggest generation of a "quantum" of second messenger by each activated thrombin receptor before shut-off, which continues even in the presence of more cleaved/activated receptor. Graded responses to thrombin appear to be generated from a balance of receptor activation rate and second messenger clearance. Notably, this hypothesis indicates that an antagonist of the thrombin receptor must only slow down the rate of receptor activation in order to block signaling.
Thrombin receptor is expressed in many different cell types including platelets, endothelial cells, smooth muscle cells, osteoblasts, fibroblasts, lymphocytes, neurons, and astrocytes.